KR0142084B1 - Method for separating keto-2l-gulonic acid from fermentation liquor - Google Patents

Abstract

Process for separating 2-keto-L-gulonic acid from a fermentation liquor chiefly containing the calcium salt of 2-keto-L-gulonic acid, by carrying out the following successive operations: 1) separation of the insoluble matter, 2) demineralisation of the filtered liquor, and 3) separation of the 2-keto-L-gulonic acid.

Description

Method for separating 2-keto-gulonic acid from fermentation medium

The present invention relates to a method for separating 2-keto-L-gulonic acid from fermentation medium. 2-keto-gulonic acid, a synthetic intermediate of ascorbic acid, is generally produced in the form of calcium 2-keto-gulonate in a fermented medium by a suitable microorganism.

In order to convert it to ascorbic acid, it is especially possible to use 2-keto-L-gulonic acid in the free form (2KLG-H) or in the form of sodium salt (2KLG-Na) in any case as pure as possible. It is advantageous.

According to Japanese patent applications JP 52.66684 and JP 53.62894, it is known to prepare substantially pure sodium 2-keto-L-gulonate from a fermentation medium containing calcium 2-keto-L-gulonate. However, industrial implementation of these processes does not allow to directly obtain the 2-keto-L-gulonic acid required for subsequent conversion to ascorbic acid.

A simple and easy regeneration process has shown that substantially pure 2-keto-L-gulonic acid (2KLG-H) can be obtained in fermentation medium containing calcium 2-keto-L-gulonate in excellent yield. This makes the subject matter of the present invention.

Fermentation medium generally contains insoluble biomass and organic and inorganic impurities in addition to 2-keto-L-gulonic acid in the form of calcium salts, which inorganic impurities are essentially associated with cations such as sodium, potassium or magnesium ions. It consists of inorganic anions.

The process of the present invention consists essentially of the following steps:

a) separating the insoluble material from the fermentation medium;

This step is by agglomeration with a flocculant, for example in the form of a polyacrylamide, followed by centrifugation;

-Preferably by coagulation under a reduced pressure with a flocculant such as polyacrylamide, for example by filtration after addition of a filtration additive such as wood powder or diatomaceous earth,

Or by ultrafiltration through a suitable inorganic or organic film such as a polyvinylidene difluoride film or a ZrO 2 film on a carbon matrix;

b) The medium from which biomass and other insolubles have been removed is evaporated under reduced pressure up to 60 to a volume corresponding to an initial volume of 1/4 to 1/3, or until about half of the volume is reached. Concentrated by reverse osmosis on a polysulfone membrane;

c) At temperatures of 40 ° or below, the inorganic cations are removed from the concentrated medium by the addition of a substantially stoichiometric amount of concentrated sulfuric acid to the calcium present. Precipitated calcium sulfate is separated by filtration and washed with water;

d) contacting a cation exchange resin with a deionized and acidified filtrate containing various cations, mostly non-precipitated calcium and sodium, potassium and magnesium cations, or preferably in the form of an acid (hydrogen) polymer cation exchange resin Cations are removed by passing through a column of (preferably sulfone type);

It is also possible to decationic and acidify the medium by direct passage in acid form, preferably onto sulfone type cationic resin, before concentration;

e) the precipitates and cations are removed and optionally concentrated medium in contact with the weak base anion exchange resin in the form of a base (hydroxyl), for example by passing a polymer anion-exchange resin of preferably dialkylamino type, thereby increasing the strength in the solution. Removes anions of inorganic acids;

f) The demineralized solution is then concentrated by evaporation under reduced pressure at up to 70% of 60% of the initial volume;

g) further concentrated under reduced pressure at about 40 ° C. until the volume is reduced to about 30-40%, followed by crystallization of the 2-keto-L-gulonic acid (optionally cooling the crystal slurry), or 20 2-keto-L-gulonic acid by extracting a concentrated and demineralized medium with a suitable organic solvent which is an optionally halogenated aliphatic or aromatic hydrocarbon containing aliphatic amine (preferably secondary) containing at least carbon atoms in the solution. Isolate. The organic solution thus obtained can be extracted with an aqueous solution of a strong inorganic acid selected from hydrochloric acid, sulfuric acid or nitric acid, the concentration of which can be 0.5N to 3N.

It may be particularly advantageous to carry out the crystallization continuously in a crystallizer which is operated by evaporation under reduced pressure at about 40 ° C. and provided with an external exchanger. The obtained crystals are separated by filtration or centrifugation and washed with water. 2-keto-L-gulonic acid is separated in the form of a monohydrate, the purity of which is generally about 100%. Hydrated water molecules can be removed by heating under reduced pressure.

In general, drying of the wet monohydrated 2-keto-L-gulonic acid crystals is carried out by transferring them under a warm air stream.

The aqueous solution thus obtained is concentrated to dryness to yield monohydrated 2-keto-L-gulonic acid in powder form, the purity of which is generally at least about 90%.

This process can be carried out continuously or discontinuously. Generally, biomass and other insoluble materials comprise 1.5 to 3 weight percent of the total medium.

The following examples illustrate the invention.

Example 1

Isolation of Insoluble Matter from Fermentation Medium

Fermentation obtained by culturing Corynebacterium strains that produce 2-keto-L-gulonic acid containing about 1.6% biomass and other insolubles and 10% calcium 2-keto-L-gulonate Use the medium.

Biomass and other insoluble materials can be separated by one of the following steps.

1) A fermentation broth (123 kg) containing sediment (about 2 kg) is passed through a Carrbosep ultrafiltration membrane having a porosity of 80,000 Da at 40 ° C. This gives a retentate (112 kg) free of insoluble product and the precipitate as a whole and a retentate (11 kg) in which the calcium 2-keto-L-gulonate concentration in it is equal to the concentration in the permeate. .

By diafiltration, the retentate is consumed by continuously adding water to the retentate in the ultrafiltration process as described above. Diafiltration is stopped when the content of calcium 2-keto-L-gulonate in the collected permeate corresponds to a recovery level of at least 99%.

2) Add an aqueous 5 g / liter solution (20 cc) of polyacrylamide-type flocculant Flouerger 8850 to the whole medium (500 g) and stir vigorously. After a few minutes of contact, the powder (10 g) is added while stirring vigorously. The resulting mixture is filtered through a filter cloth under reduced pressure (300 mmHg; 40 KPa). The filter cake is washed with water (50 cc).

The loss of calcium 2-keto-L-gulonate is 2% or less. The weight of the filtrate containing 9.4% of calcium 2-keto-L-gulonate is 520 g.

3) Agglomerated whole medium (200 liters) containing 2.7% precipitate with 5 g / liter aqueous solution (8 liters) of polyacrylamide-type flocculant (Floirzer 8850) and then reinforced plate-centrifugation with a surface area of 7200 m 2 It is continuously introduced at a flow rate of 2000 liters / hour. Recover the clarified medium containing 0.1% precipitate.

Example 2

Concentration of medium

The medium from which the insoluble material has been removed can be concentrated according to one of the following methods:

1) The medium (225 liters) without insoluble matter is concentrated by evaporation under reduced pressure (72 mmHg; 9.5 KPa) at 47 ° C. to 62 liters. The heat reduction is 0.5% or less.

2) 30 liters of insoluble material-free medium (60 liters) through a reverse osmosis module equipped with a polysulfone membrane (PCI Z 99) at a flow rate of 25 liters / h. Concentrate by passing until a volume of is obtained.

Example 3

Precipitation of Calcium Sulfate

In a stirred reactor containing the concentrated medium (2 liters) obtained under the conditions described in Example 2 1) and maintained at 40 ° C. or lower, corresponds to the total amount of calcium present in the medium (moles ( mole)) add 100 cc of concentrated sulfuric acid. Calcium sulfate precipitated in the form of this anhydride is separated by filtration and washed with water. The filtrate and collected washes contain 99.5 mol% of initial 2-keto-L-gulonic acid. Calcium removal yield is 95%.

Example 4

Removal of cations

3 liters of the filtrate obtained in Example 3 (before mixing with water in the washing of the calcium sulfate cake) was 80 cm high containing a strong cation resin (1.6 liters; Amberlite IRC120) in hydrogen form, Pass through a column of 5 cm diameter.

After washing, a medium (5.5 liters) is obtained with a sulfate solids content of 1% or less relative to 2-keto-L-gulonic acid present in the medium.

The recovery level of 2-keto-L-gulonic acid is at least 99.5%.

Example 5

Removal of negative ions

Medium obtained in Example 4 through a 10 cm high, 1.4 cm diameter column containing weak anion exchange resin (15 cc; Amberlite IRA93) in the form of hard siloxane to reduce the sulfuric acid content in the medium acidified by Factor 10 Pass (0.4 liters). The loss of 2-keto-L-gulonic acid does not exceed 2% of the amount introduced.

Example 6

Crystallization of 2-keto-L-gulonic Acid

The medium (5000 g) obtained under the conditions of Example 5 was concentrated by evaporation to give a solution (3430 g) containing 2-keto-L-gulonic acid (1170 g) and impurities (225 g). The concentrate thus obtained is partially concentrated at 40 ° C. under reduced pressure (40 mmHg; 5.3 KPa) until a weight of 2010 g is reached. This further concentration leads to crystallization of the hydrated 2-keto-L-gulonic acid. The crystal slurry is cooled to 25 ° C. The crystals are separated by filtration and washed with water. Hydrated 2-keto-L-gulonic acid (930 g) is obtained, the purity of which is at least 99% and its sulfate solids content is below 0.1%. Yield 73%.

The filtered mother liquor and wash are collected and concentrated to 710 g under reduced pressure (40 mmHg; 5.3 KPa). After cooling the crystals are separated by filtration and washed with water. This gives monohydrated 2-keto-L-gulonic acid (285 g) with 89% purity.

Wet (5.8% water) crystals (9400 g) of 2-keto-L-gulonic acid monohydrate having a purity of 99% or more are dried in air driers with air circulating at 5 ° C. at 75 ° C. for a residence time. Is 3 seconds. This yields monohydrated 2-keto-L-gulonic acid (8850 g). It can be dehydrated by heating it to 40 ° C. for several hours under reduced pressure (5 mmHg; 0.67 KPa).

Example 7

A medium containing 2-keto-L-gulonic acid (80 g) having a purity of 84% and free of precipitates and cations (1 liter) was subjected to agenogen 83 (Adogen 83, Schering) in kerosene at 50 ° C. for 0.5 hour. 260 g) of a solution (1 liter). 2-keto-L-gulonic acid (83%) extracted into the organic phase is quantitatively re-extracted with 1N aqueous nitric acid solution (690 cc).

After concentration to dryness, the solution provides a crystallized product (81.5 g) containing monohydrated 2-keto-L-gulonic acid (89%).

Claims (1)

a- insoluble material is separated from the fermentation medium by ultrafiltration or filtration in the presence of a flocculant and a filtration additive or by centrifugation in the presence of a flocculant and b- evaporated under reduced pressure or obtained by reverse osmosis. The mixture is concentrated and c- stoichiometric amounts of sulfuric acid are added to the mixture to precipitate almost all of the calcium sulfate to remove inorganic cations from the concentrated mixture, then to separate the calcium sulfate, and the d- filtrate is polymerized in an acid cycle. Contact with the cation exchange resin to remove the cation, e- filtrate is contacted with the polymer anion exchange resin to remove residual strong acid ions, concentrate the mixture under f- decompression, and further crystallization of the solution under g- decompression 2-keto-L-gulonic acid, or aliphatic amine containing 20 or more atoms in solution Extracting with an organic solvent selected from halogenated or non-aliphatic or aromatic hydrocarbons, followed by extraction with aqueous hydrochloric acid, nitric acid, or sulfuric acid solution, and then isolated 2-keto-L-gulonic acid after dry concentration of the aqueous solution obtained. A method for producing 2-keto-L-gulonic acid from a fermentation medium containing a calcium salt of 2-keto-L-gulonic acid.